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Evidence based theory for integrated genome
regulation in ontogeny
- potential therapeutic utility
Michal K. Stachowiak Ewa K. Stachowiak Molecular and Structural Neurobiology and Gene Therapy Program State University of New York
At Buffalo
Paul Davies: “Scientists and clinicians who study cancer …….are caught up in the frantic and expensive search for an elusive "cure" but rarely ask why these diseases exist at all and what their place is in the grand story of life on Earth” Physics of Cancer: New tools and fresh perspectives, Physics World Vol. 26 No. 7, 2013, 37--‐40.
Unicellular
Oncogenes: P53, cdk, klf4, Ddk, cyclins, ...
- 3 billion years
Unicellular
Multicellular
Oncogenes: P53, cdk, klf4, Ddk, cyclins, ...
Morphogenes: Hox, Wnt, Notch, Pax, Rest, mesp2, … Oncogenes: P53, cdk, klf4, Ddk, cyclins, ….
- 3 bilion years
- 0.6 bilion years
{ }
Unicellular
Multicellular
Oncogenes: P53, cdk, klf4, Ddk, cyclins, ...
Morphogenes: Hox, Wnt, Notch, Pax, Rest, mesp2, … Oncogenes: P53, cdk, klf4, Ddk, cyclins, ….
- 3 bilion years
- 0.6 bilion years
{ }
Integrative Nuclear FGF-2 + FGFR1 (F-F) Signaling - INFS
Stachowiak, M.K., X. Fang, J.M. Myers, S.M. Dunham, R. Berezney, P.A. Maher, and E.K. Stachowiak. 2003b. Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiation. J Cell Biochem. 90:662-691; DNA & Cell Biology, 2007).
…thousands of genes…. NATURE
NURTURE
INFS
Evolution of FGFs & FGF Receptors as nuclear signaling
proteins
Stachowiak et all, 2015
FGF general structure: FGF cellular action:
Unusual properties of FGFR1 transmembrane domain
Chou-Fasman and Garnier algorithms predict the TM of FGFR4 to be a typical a-helix. In contrast FGFR1 showed a b-sheet-Turn-b-sheet structure with no predicted a-
helix. Both FGFR2 and FGFR3 demonstrated b-sheets followed by shorter a-helices. (= -helics; = -sheet; T= turn, C=random coil). (Myers et al. 2001)
TM
R1
R1
R1
R1
R1
RSK
R1
ER
R1
R1
Golgi Apparatus
cis
medial
trans
R1
R1
Nucleus Cell Surface
RSK
Importinβ
FGF2
FGF2
R1
Nuclear FGFR1 (R1) trafficking by RSK1, FGF & Importin
Stachowiak, M.K. Stachowiak E.K. DNA and Cell Biology 2007; Dunham, Prasad, Stachowiak, J. Biophys. 2006; Reilly, J.F., and P.A. Maher. 2001. Importin beta-mediated nuclear import of fibroblast growth factor receptor: role in cell proliferation. J Cell Biol. 152:1307-1312 Bryant, D.M., Wylie, F.G., and Stow, J.L. (2005). Mol Biol Cell 16, 14-23. . )
R1
“Integrative Nuclear FGFR1 Signaling (INFS)”
R1
R1
R1
R1
R1
RSK
R1
ER
R1
R1
Golgi Apparatus
cis
medial
trans
R1
R1
Nucleus Cell Surface
RSK
Importinβ
FGF2
FGF2
R1
Ca++ cAMP RAR PY
Nurs
NGF
BMP7
RA
Ach
Ang
Vit D
Stachowiak, M.K. Stachowiak E.K. DNA and Cell Biology 2007; Stachowiak,M.K., Stachowiak,E.K., Aletta, J.M. and Tzanakakis,E.S; In: Stem Cells from Mechanisms to Technologies, M.K. Stachowiak, E.S.
Tzananakis-editors. World Scientific Publishing, 2011
R1
Knock out prevents gastrulation
FGFR1 is indispensable for ontogeny
FGFR1 +/+ FGFR1 -/-
(Gene @ Dev., 8, 3045-57, 1994)
Yabut & Bernstein; Aging Volume 3, No. 5 2005
INFS in Pluripotent Embryonic Stem Cells
Yabut & Bernstein; Aging Volume 3, No. 5 2005
INFS in Pluripotent Embryonic Stem Cells
All trans - Retinoic Acid (RA)
FGFR1 Nuclear
Accumulation
mESC
0 hr 48 hr 0 hr 48 hr
hESC
hNPC
0 hr 24 hr
hNB Cells
Lee, Y-W., Terranova, C., Birkaya, B., Narla, S., Kehoe, D., Parikh,P., Dong, S., Ratzka, A., Brinkmann, H., Aletta, J., Tzanakakis, E., Stachowiak, E.K., Claus, P., and Stachowiak, M.K. (2012). A novel
nuclear FGF Receptor-1 partnership with retinoid and Nur receptors during developmental gene programming of embryonic stem cells J. Cell. Biochem., 113, 2920-2930
48 hr 0 hr
Nuclear Accumulation of FGFR1 is a common response to Retinoic Acid (RA) in Various Cells
nFGFR1 mediates RA-induced neuronal programing of pluripotent mESC
Lee, Y-Wet al., (2012). A novel nuclear FGF Receptor-1 partnership with retinoid and Nur receptors during developmental gene programming of embryonic stem cells J. Cell. Biochem., 113, 2920-2930
FGFR1 localizes within transcription domains
RAR RXR
CBP CREB Nur
FGFR1
In multiprotein CBP complexes:
FGFR1 RNA Polymerase II
Structured-Illumination Microscopy (SIM)
Insight into the whole genome !
Illumina HiSeq 2000
Illumina HiSeq 2000
cDNA synthesis
sequencing
Cross-linked Chromatin Immunoprecipitation,
DNA sequencing (ChIPseq)
RNA
RNA and small RNA sequencing
(RNAseq)
Global developmental gene programing involves a nuclear form of Fibroblast Growth Factor Receptor-1 (FGFR1). 2015, PLOS One, in press
Integrative paradigm for global developmental genome programing in embryonic stem cells by nuclear Fibroblast Growth Factor Receptor-1 (FGFR1) ISSCR, 2015
Utilizing induced Pluripotent Stem Cells (iPSCs) to delineate the neurodevelomental base of schizophrenia, ISSCR 2015.
Lessons from ChIPseq:
(1) nFGFR1 and its partners RXR and Nur70 target all chromosomes
(2) nFGFR1 genome binding is more frequent in NC (3) nFGFR1 targets genome alone as well as with RXR and Nur77
promoter gene body intergenic
-5 -1 TSS
Binding sites enrichment: 10x 10-100x 3-5x 0.8x
(4) In mESC and NC genomes 2/3 of targeted sites are within promoter
and genic regions
(-)5kb to (-)1kb TSS, 466
(-/+)1kb TSS, 3396
Genic Region, 3772
Intergenic, 3744
(-)5kb to (-)1kb TSS, 1980
(-/+)1kb TSS, 14270
Genic Region, 17578
Intergenic, 12309
FGFR1 in ESC FGFR1 in NC
Genes: 3658
Genes: 12780
(5) nFGFR1, RXR and Nur77 bind predominantly to proximal promoters
of expressed genes
(ChIPseq+RNAseq)
Ingenuity Pathway Analysis (IPA): Combination of ChIP- and RNA-seq
Genomic DNA
-/+ 1kb TSS
nFGFR1
*
In ESC nFGFR1 controls Pluripotency transcriptional network by targeting
Suz12
Genes bound in pluripotent ESC Genes upregulated in ESC *
(OSKM) Oct4, Sox2, Klf4, Myc
network
nFGFR1
nFGFR1
nFGFR1
*
*
*
*
nFGFR1
* *
*
*
Genes bound in pluripotent ESC Genes bound in RA differentiated ENC Genes upregulated in NC Genes downregulated in NC *
*
*
*
nFGFR1 turns off pluripotency transcriptional network during Retinoic
Acid (RA) induced ESC differentiation
nFGFR1 targets genes of the cell proliferation and survival pathways
Top biological functions, networks and diseases: Top network controlling cell proliferation and survival:
Building body parts and axes: nFGFR1 mediates RA activation of the Homeobox genes (Hox clusters genes)
RED: UPREGULATED GREEN: DOWNREGULATED
Homeobox (Hox) genes are critical for organism development and patterning
- including CNS
Nuclear FGFR1 binding to 3’ Hoxa gene cluster in the presence of RA
Dominant negative nuclear FGFR1 (NLSTK-) blocks RA upregulation of core
3’ Hoxa genes
Nuclear active nuclear FGFR1 (NLS) upregulates core 3’ Hoxa genes
in the absence of RA
Change in the Hierarchy of Master Genes
lower rank master genes
FGFR1 Hox Further
“Grand master” down-stream genes
Further down-stream genes
Old New
Hox “Grand master”
lower rank master genes
Decisions – brains or muscles?
nFGFR1 upregulates neuronal and downregulates mesodermal genes:
Neuronal genes:
Mesodermal genes:
Building Brains - nFGFR1 targets:
A
Activated Wnt/β-catenin signaling pathways in NCs
Building Brains - nFGFR1 targets:
“CREB signaling in neurons pathway”
“Axonal guidance signaling canonical pathway”
active chromatin
hub
FGFR1
gene1 gene2
gene3
active chromatin
hub
FGFR1
gene1 gene2
gene3
Chromatin
Conformation
Capture –
6C analysis
How to coordinate regulation of the thousands of genes?
Role of multi-gene chromatin hubs
“Transcriptional factories”:
RA #14 FGFR1 –Alexa 488
Chromosome 6-- 5’ Alexa Fluor 546
chromosome6-- 14’ Alexa Fluor 546
MERGED DAPI
In Situ Hybridization/Immunocytochemistry verifies interchromosomal interactions
involving nFGFR1
FGFR1 Chr.6 Chr.14
active chromatin hub
FGFR1
gene1 gene2 gene3
FGFR1
gene1 gene2
Interactions of pde1c (red arches) and hoxA1 (orange arches).
Looping interactions within intra-chromosomal (Chr. 6) loci:
active chromatin
hub
FGFR1
Gene 1
Gene 2 Gene3
FGFR1 and RNA Polymerase II in shared nuclear domains
0.4 – 2.0 um speckle
nFGFR1 forms multi-gene chromatin hubs
“Transcriptional factories”:
Evidence based theory of ontogeny –
Nuclear FGFR1 a new type master gene regulator
Global developmental gene programing involves a nuclear form of Fibroblast Growth Factor Receptor-1 (FGFR1). 2015, PLOS One, in press
“Therapeutic”Targeting of
INFS
Rat pheochromocytoma (PC12)
(INFS+ cells)
differentiates cancer cells
New neurons in adult brain
PNAS US A. 2005 02(32):11539-44; Integrative Biology, 2009, 1, 394 – 403. Stem cells translational medicine 2013, 2:776-788.
Mol. Biol. Cell, 2009, 20, 2401 – 2412.
J. Biol. Chem. – 280(31):28451-62.
PloS one. 2013, 8:e68931
Michal and Ewa’s labS: Barbara Birkaya Yu-Wei (Eli) Lee Christopher Terrranova Sridhar Narla Nived Nair David Freedman Courtney Benson Seerat Elahi Brendon Decker .
Collaborators: John Aletta H3 Biosystems Merouanne Bencherif (Targacept Inc.) Ronald Berezney (UB) Dhruba Bharali (UB Dr. Prasad lab) Michael Buck (UB) Peter Claus (Hannover Med. Sch., Germany) Janusz Morys, Ilona Klejbor (Med. Univ. Gdansk, Poland Hari Shroff (NIH) Manolis Tzanakakis (Tufts Univ,)
Support: NIH (3), NSF (3), NYSTEM(2), Patrick Lee Foundation & Esther Trachtman Foundation for Schizophrenia, National Parkinson Foundation, Rett Foundation, Targacept Inc..
State University of New York
At Buffalo
The End